In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological samples are preserved from decay. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated samples. The purpose of fixation is to preserve a sample of biological material as close to its natural state as possible. Fixed samples are used for examination or analysis.
Fixatives can be classified as cross-linking or precipitating fixatives.
Cross-linking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. Aldehydes are by far the most commonly used cross-linking fixatives. Although aldehyde-fixed biological samples are useful for histological, pathological, and cell biological applications, they pose several problems for molecular analysis of the preserved sample. For example, fixation with aldehydes causes protein-protein, DNA-protein, and RNA-protein cross-links to form, which interferes with the ability to extract and purify proteins and nucleic acids. Moreover, reversal of cross-linking often results in free aldehyde in the sample, which can interfere with functional proteins (such as enzymes or antibodies), nucleic acid probes, resins, or any other functional reagents with amino groups that are used in sample processing and analysis.
As such, there remains a need for methods and compositions that increase the efficiency of isolating various components (such as nucleic acids, proteins, and organelles) from biological samples fixed in fixed in aldehyde-based cytology media.
Precipitating fixatives act by reducing the solubility of protein molecules and disrupting hydrophobic interactions. As this process is very different from cross-linking fixation, biological samples fixed with precipitating fixatives often must be processed with different reagents and methods than those used with cross-linking fixatives. Alcohols are commonly used precipitating fixatives. There is a need for methods and compositions that increase the efficiency of isolating various components (such as nucleic acids, proteins, and organelles) from biological samples fixed in alcohol-based cytology media.
Therefore, there remains is a need for methods and reagents that are useful in extracting various components from fixed biological samples (such as nucleic acids, proteins, and organelles), regardless of the type fixative used. In particular, lysis solutions are needed that may be used for biological samples fixed in cytology media that is cross-linking-based, precipitating-based, or both.